The long-term objectives of this research project are to understand the structure, function and evolution of mammalian genes and to reconstruct phylogenetic relationships among mammals. Our specific aims for the first three years are: (1) to understand the structure, function, and evolution of the apolipoprotein multigene family, (2) to resolve a long-standing controversy over the phylogenetic relationship among human, rat, rabbit, cow and dog, and (3) to test the molecular evolutionary clock hypothesis. The apolipoproteins are the protein components of plasma lipoproteins, which are the major carriers of various lipids in humans blood, and have been showed to participate in lipoprotein assembly, secretion, processing, and catabolism, lipoprotein assembly, secretion, processing and catabolism. Because of their medical and biological importance, it is desirable to have a detailed knowledge of the structure, structure-function relationships, and evolution of the apolipoproteins. This can best be achieved by a combination of molecular and statistical techniques. Parts of the gene for apoB-100 and the complete cDNAs of the genes for apoA-I and apoE in opossum, rat, rabbit, cow and dog will be cloned and sequenced. The new as well as published sequence data will be used to compute the rates of nucleotide substitution, to examine deletions and insertions, to infer the structure-function relationships, and to infer the evolutionary history of the apolipoproteins. It will be particularly important to see how well the putative low density lipoprotein binding regions in apoB-100 have been conserved in evolution. The data together with other sequence data will be used to infer the phylogenetic relationships among Primates (man), Rodentia (rat), Lagomorpha (rabbit), Artiodactyla (cow), and Carnivora (dog), using various phylogenetic reconstruction methods; opossum will serve as an outgroup. Sequence data from apolipoprotein genes are ideal materials for this purpose because these genes have evolved rather rapidly and do not seem to have been involved in any gene conversion events. The data will also be very useful for testing the molecular clock hypothesis, particularly with opossum as an outgroup. The study is likely to resolve the controversy over whether rates of nucleotide substitution are in general higher in short-living than in long-living mammals.